55] Lipid A 4′-kinase from Escherichia coli: Enzyme assay and preparation of 4′-32P-labeled probes of high specific radioactivity

Abstract

Publisher Summary The Escherichia coli ( E.coli) lipid A 4 ' -kinase is the enzyme responsible for the addition of the 4 ' -phosphate to the diglucosamine backbone of gram-negative lipid A. In the biosynthetic scheme of lipid A, the 4 ' -phosphoryl group is transferred from the γ-position of adenosine triphosphate (ATP) to the substrate tetraacyldisaccharide 1-monophosphate (DSMP) to yield the lipid A precursor lipid IV A . By making use of [γ- 32 P]ATP as a substrate, the action of the 4 ' -kinase can be used to generate 32 P-labeled lipid IV A of high chemical purity (98–99%) and specific radioactivity (3000 Ci/mmol). Because lipid IV A is a precursor of lipid A and is bioactive in eukaryotic cells, 32 P -labeled lipid IV A is a useful probe for the investigation of both bacterial and eukaryotic aspects of lipid A biochemistry. The chapter describes assays for measuring the activity of the 4 ' -kinase and a procedure that employs this enzyme to generate 32 P-labeled lipid IV A to be used as a labeled lipid A probe.